Endoscopic techniques have provided sensitive means of detecting adenomatous polyps that could potentially progress to colon cancer. Thus, endoscopic monitoring of susceptible individuals has made it possible to detect and remove early dysplastic lesions before they become life threatening. To date, visualization of the dysplasia has been limited to detection of anatomical abnormalities, and there is no precedence of live imaging of biological activity that is predictive of tumor growth versus regression. In this study we have shown that cathepsin B activity is an inherent component of cancer-associated inflammation in tumor infiltrating myeloid cells. We have demonstrated that live imaging of this activity is feasible, and provides for accurate demarcation of dysplasia and the associated neo-angiogenesis. Furthermore, we have demonstrated that this mode of imaging reveals dynamics of biological activity that is predictive of tumor progression versus response to therapy.
Cathepsin B activity has been associated with a number of tumors in humans and experimental animals, making it an attractive avenue for the imaging and possible treatment of cancer 
. Here we provide evidence that in addition to cathepsin B, another member of the cysteine family of cathepsin Z is significantly up regulated in adenomatous polyps. Cathepsin activities were focal and readily distinguished the dysplastic lesions from the healthy neighboring tissue. Thus, imaging of this activity using protease sensitive probes allows accurate detection of areas of dysplasia that may be undetectable by visible light imaging, due to size limits or anatomical features.
The source of cathepsin activity in tumors has been in the past debated, with some reports emphasizing that tumor cells are the major source of this activity. Here, we provide evidence based on histology and flow cytometry that clearly reveals tumor infiltrating myeloid lineage cells as the predominant source of this activity. This is a fortunate and useful finding, as cancer-associated inflammation is causatively linked with adenoma growth. Significantly, our analysis of fluorescent signals detected with in the lesions revealed that the local increase in signal intensity was due to increased numbers of cathepsin active cells rather than higher activity per cell. Furthermore, we could confidently and accurately detect this activity to a depth of at least 75 µm. Thus, the imaged signal was directly reporting the abundance of pro-inflammatory cells within the dysplasia.
We demonstrated that genetic ablation of cathepsin B results in suppression of tumor infiltrating pro-inflammatory cells, notable attenuation of polyposis, and a decrease in the fluorescent signal emanating from the lesions. Ablation of cathepsin B significantly increased the overall levels of active cathepsin Z, Previous reports suggest that cathepsin Z (also called cathepsin X) compenstates for the levels of membrane bound cathesin B, and is elevated in cathpesin B knock out mice 
. Thus, attenuation of probe signal in polyps arising in cathepsin B knock mice suggests that the probe activity was relatively specific for cathepsin B. Furthermore, attenuation of polyposis in these mice suggests a specific requirement for cathepsin B in the progressive growth of polyps.
Surprisingly, cathepsin B deficiency predominantly affected the CD11b+Gr1+ MDSC infiltrate, and did not impact the CD11b+F480+ macrophage component of polyp infiltrating leukocytes. Both of these myeloid cell types contributed equally to the local activation of the ProSense 680 probe. Since preferential loss of MDSC correlated with polyp attenuation, we conclude that these cells were critically contributing to the progressive growth of dysplasia. Furthermore, we conclude that the decrease in cathepsin activated probe signal was largely due to the loss of MDSCs from areas of dysplasia.
The cytokine TNFα is regarded to be at the apex of inflammatory responses, promoting angiogenesis, mobilization of neutrophils and escalation of inflammation 
, including cancer associated inflammatory reactions 
. Treatment of mice with anti-TNFα suppresses pathogen induced inflammatory bowel disease and inflammation triggered cancer 
. We therefore postulated that if pro-inflammatory cells were the source of cathepsin activity and played a causative role in polyposis, then suppression of polyposis-associated inflammation should hinder progressive polyp growth, and this therapeutic effect should be reflected in a significant down-regulation of cathepsin activity. Treatment of mice with anti-TNFα resulted in a preferential loss of MDSC infiltrating the lesion, in increased apoptosis of the aberrant epithelial cells, and regression of the lesions. Accordingly, cathepsin-B activities as measured by western blot analysis and probe signal were significantly attenuated. Altogether, these observations establish live imaging of cathepsin B activity with sensitive near infrared probes as a highly specific method for detection of biological activity linked with progressive tumor growth.
Tumor associated neo-angiogenesis is considered to be a necessary pathological component of tumor growth and a viable target for therapeutic intervention. Pro-inflammatory cells are a recognized source of angiogenic factors, and suppression of inflammation is therefore expected to impact tumor associated inflammation. It is not known at what stage dysplasia triggers angiogenesis and how the dynamics of infiltrating leukocytes reflect the expansion or regression of blood vessels in the lesion. Here, taking advantage of a constitutively active near infrared probe we have imaged in a living animal the neo-vascularization of early dysplastic lesions by micro-vessels, and revealed the change in architecture of the vasculature entering the lesion. Using different fluorescent excitation and spectral separation of the images we succeeded in simultaneously imaging both angiogenesis and cathepsin B activity in the same tissue and 3-dimensional space. The visualization of the architecture of the lesion was further enhanced by concomitant imaging of auto-fluorescent signals that emanated largely from the tumor mucosa
We were able to demonstrate that anti-inflammatory regiments such as ablation of cathepsin B or treatment of mice with anti-TNFα impacts the infiltration of dysplastic lesions by the microvasculature, and that loss of the vessels correlates with attenuation or regression of the lesions. Thus, near infrared imaging of blood microvasculature provides an independent means of monitoring progressive dysplasia, and response to effective therapeutic intervention.
In summary, we have shown that live imaging of mechanism-based near infrared probes allow simultaneous detection of independent biological activities that report progressive tumor growth. Our earlier studies have documented the feasibility of application of this mode of imaging to endoscopic monitoring of cancerous lesions. Here, we have provided mechanistic information on the source and biological significance of the signal. All together, we have documented a powerful advance in our visualization of tumor biology dynamics, which allows for sensitive monitoring cancer progression or favorable response to therapy.